The present invention is concerned with an IBDV mutant comprising the nucleotide sequences encoding a variant E VP2 protein in segment A of the viral genome and that is capable to infect and replicate in CEF tissue culture, a vaccine comprising this mutant and the use of this mutant for the manufacture of a vaccine.
Infectious bursal disease virus (IBDV) is a member of the Birnaviridae family. Viruses in this family have a very similar genomic organisation and a similar replication cycle. The genomes of these viruses consist of 2 segments (A and B) of double-stranded (ds) RNA. The larger segment A encodes a polyprotein, which is cleaved by autoproteolysis to form mature viral proteins VP2, VP3 and VP4. VP2 and VP3 are the major structural proteins of the virion. VP2 is the major host-protective immunogen of birnaviruses, and contains the antigenic regions responsible for the induction of neutralising antibodies. The VP4 protein is the virus-coded protease that is involved in the processing of a precursor polyprotein of the VP2, VP3 and VP4 proteins. The larger segment A possesses also a second open reading frame (ORF), preceding and partially overlapping the polyprotein gene. This second open reading frame encodes a protein VP5 of unknown function that is present in IBDV infected cells. The smaller segment B encodes VP1, a 90 kDa multifunctional protein with polymerase and capping enzyme activities.
For IBDV, two serotypes exist, serotype 1 and 2. The two serotypes may be differentiated by virus neutralisation (VN) tests. Furthermore, subtypes of serotype 1 have been isolated. These so-called xe2x80x9cvariantxe2x80x9d viruses of serotype 1 can be identified by cross-neutralisation tests, a panel of monoclonal antibodies or RT-PCR. These subtypes of serotype 1 of IBDV have also been described in literature, for example: classical, variant-E, GLS, RS593 and DS326 strains (Van Loon, et al. Proceedings of the International symposium on infectious bursal disease and chicken infectious anaemia, Rauischholzhausen, Germany, 179-187, 1994).
Infectious Bursal disease (IBD), also called Gumboro disease, is an acute, highly-contagious viral infection in chickens that has lymphoid tissue as its primary target with a selective tropism for cells of the bursa of Fabricius. The morbidity rate in susceptible flocks is high, with rapid weight loss and moderate mortality rates. Chicks that recover from the disease may have immune deficiencies because of the destruction of the bursa of Fabricius, which is essential to the defence mechanism of the chicken. The IBD-virus causes severe immunosuppression in chickens younger than 3 weeks of age and induces bursal lesions in chicks up to 3 months old.
For many years the disease could be prevented by inducing high levels of antibodies in breeder flocks by the application of an inactivated vaccine, to chickens that had been primed with attenuated live IBDV vaccine. This has kept economic losses caused by IBD to a minimum. Maternal antibodies in chickens derived from vaccinated breeders prevent early infection with IBDV and diminish problems associated with immunosuppression. In addition, attenuated live vaccines have also been used successfully in commercial chicken flocks after maternal antibodies had declined.
Recently, very virulent strains of IBDV have caused outbreaks of disease with high mortality in Europe. The current vaccination programs failed to protect chicks sufficiently. Vaccination failures were mainly due to the inability of live vaccines to infect the birds before challenge with virulent field virus.
A further cause of acute disease in vaccinated flocks is the emerging of antigenic variants in the mid-1980s, in particular in the USA. The most important new antigenic subtypes of serotype 1 IBDVs were the Delaware variant E and GLS strains. Until then, vaccines used were based on live attenuated or killed IBDV strains of the classical type only. However, despite the fact that classical IBDV vaccine strains induce a certain degree of cross-protection against infection of chickens by other subtype strains (and vice-versa), substantial economic losses have been sustained as a result of vaccination with vaccines based on only one of the subtype viruses.
As a result of these developments in the field it became necessary to incorporate both classical and variant IBDV subtype vaccine strains into the IBDV vaccines to obtain a broad spectrum protection.
As described above, both live and killed vaccines based on classical strains are generally used in the field. The killed classical vaccines are commonly administered per injection, whereas the live classical vaccines are administered per injection or by one of the inexpensive, mass application techniques, such as spray-, aerosol and drinking water vaccination.
Delaware variant E viruses identified so far are highly virulent and can be administered as an inactivated vaccine only. Until now, there has been only one successful attempt to adapt and attenuate a variant E strain in cell culture. This vaccine strain designated as 89/03 is able to induce protection against variant E infection, but has the disadvantage that it does not take if administered by one of the mass application routes. This vaccine virus needs to be administered by injection, subcutaneously or intramuscularly (Hein et al., Proceedings of the 43th Western poultry disease conference, pages 102-103, 1994, Sacramento, USA).
For protecting birds against infection by IBDV of the GLS subtype, only inactivated vaccines are available.
Mundt (J. Gen. Virology 80, 2067-2076, 1999) identified the structural requirements at the molecular level which allows IBDV strains that were only able to infect and replicate in vivo in the bursa of Fabricius, to infect and replicate in chicken embryo fibroblast (CEF) tissue culture as well. Two amino acid exchanges in the VP2 protein at positions 253 (Gln to His) and 284 (Ala to Thr) were necessary and sufficient for the ability of IBDV bursa strains (BU) to infect CEF and other tissue cultures. In particular, two chimeric D78/variant E IBDV mutants with a tissue culture (TC) phenotype are disclosed in this document.
In view of the above, a need existed for a live attenuated Delaware variant E IBDV vaccine that is efficacious if administered via a mass application route. Furthermore, a need existed to obtain a broad spectrum IBDV vaccine strain that affords adequate protection against IBDV strains of two or more subtypes. Such a broad spectrum vaccine prevents that separate vaccine viruses for each IBDV subtypes have to be manufactured and formulated into a combination vaccine.
It is an object of the present invention to provide an IBDV mutant that is able to induce a protective immunity in chickens against virulent strains of at least two IBDV subtypes. It is a further object to provide an IBDV mutant which affords protection against virulent IBDV variant E strains and that at the same time can be administered via a mass application technique commonly used for live IBDV vaccination.
It has been found now that these objects have been met by providing an IBDV mutant comprising the nucleotide sequences encoding a variant E VP2 protein in segment A of the viral genome and that is capable to infect and replicate in CEF tissue culture, characterised in that the mutant comprises one or more mutations in the variant E VP2 coding region such that,
(i) the codon for the amino acid at position 254 encodes Glycine, and/or
(ii) the codon for the amino acid at position 270 encodes Threonine.
It has been shown by the present inventors that the chimeric IBDV D78/variant E bursa (BU) mutant disclosed in Mundt et al. (1999, supra), displaying the culture (BU) and immunogenicity characteristics of a variant E strain, is virulent and induces complete lymphocyte depletion after administration to chickens. Furthermore, it is shown in Example 2 that the corresponding tissue culture (TC) mutant of this strain is less virulent and is able to induce a partial protective immune response if administered per injection. However, at the same time the TC mutant induces only poor protection against challenge if this mutant is administered via the ocular route (a route that is a model for drinking water administration). Surprisingly, the inventors have identified that further mutations in the naturally occurring codons for amino acids 254(Ser) and/or 270(Ala) in the VP2 gene of variant E viruses, resulting in codons encoding amino acid 254(Gly) and/or 270(Thr), provide a live attenuated IBDV mutant that:
(i) is able to induce virus neutralising (VN) antibody titers against classical- and variant E subtype IBDV strains of about the same level,
(ii) is able to induce protection in vaccinated animals against challenge with virulent classical- and variant E subtype IBDV strains,
(iii) is able to induce protection if administered via a mass application route.
IBDV subtypes are well defined in the art, e.g. by means of their reaction pattern with monoclonal antibodies. An IBDV of the variant E subtype specifically reacts with virus neutralising Moab 67 that has been deposited at the ATCC by Dave Snyder on Sep. 14, 1992, under accession no. HB 11122 (see also, Vakharia et al., Virus Research 31, 265-273, 1994).
The mutations resulting in an IBDV mutant according to the invention are introduced in the coding region for the variant E VP2 protein at the codons for amino acid 254(Ser) and/or 270(Ala).
An advantageous IBDV mutant according to the invention comprises the desired mutations in both codons or in the codon for amino acid 254.
The exchange of nucleotide 890 (A to G) and 938 (G to A) lead to the substitutions of amino acid 254 (Ser to Gly) and 270 (Ala to Thr), respectively. As described above, the genomic organisation of IBD viruses is well established: the IBDV genome comprises a large segment A and a smaller segment B. The segment A of IBDV comprises a large open reading frame (ORF) encoding a polyprotein of about 110 kDa (VP2-VP4-VP3; FIG. 1). The complete nucleotide sequences of the segment A and segment B of many IBDV strains have been determined. Furthermore, the location within the ORF, the nucleotide sequence encoding the variant E VP2 protein and the amino acid sequence of the variant E VP2 protein have been determined by Vakharia et al., Avian Diseases 36, 736-743, 1992; Vakharia et al., Virus Res. 31, 265-273, 1994; Heine et al., J. Gen. Virol. 22, 1835-1843, 1991. Additionally, the nucleotide sequence of the DNA fragment comprising variant E VP2 coding sequences (nucleotides 647-1725) and corresponding to amino acid sequences 172-532 of the polyprotein used for generating an IBDV mutant according to the invention as described in the Examples are shown in SEQ ID. No. 1 and 2.
The numbers used herein to indicate the amino acid positions refer to numbering of the amino acids in the IBDV polyprotein as commonly used in the art. The numbers indicating the nucleotide positions are based on the complete nucleotide sequence of the segment A of the IBDV genome as described by Mundt and Mxc3xcller (J. Gen. Virol. 77, 437-443, 1995; NCBI accession number X 84034).
It is demonstrated in Mundt (1999, supra) that the prerequisite for an IBDV strain with a classical- or variant E phenotype to infect and replicate in CEF tissue culture is the presence of codons encoding amino acid 253(His) and 284(Thr) in the VP2 gene of the respective strains. Therefore, in particular, an IBDV strain according to the present invention displaying the property to infect and replicate in CEF tissue culture comprises amino acid 253(His) and 284(Thr). Such an IBDV strain can be obtained by the methods disclosed in Mundt (1999, supra). Furthermore, such a method is also described in Example 1.
As a result of the degeneracy of the genetic code various possibilities exist for the codons encoding amino acid 254(Gly) and 270(Thr) of the polyprotein of an IBDV mutant according to the invention. The codon in the VP2 coding region for amino acid 254(Gly) may be GGA, GGC, GGG or GGT, GGC being preferred, whereas the codon for amino acid 270(Thr) may be ACT, ACC, ACA or ACG, ACG being preferred.
Variant E IBDV strains having the property (i) to infect and replicate in CEF tissue culture and (ii) to induce a broad-spectrum immune response upon administration via a mass application route, can be prepared by introducing the specific relevant codons mentioned above in a naturally occurring variant E strain.
Alternatively, this result can also be obtained by exchanging part of the variant E VP2 coding region by a corresponding part of the genomic sequence of a known (classical) IBDV strain that already comprises the desired codons at the relevant positions. Full length genomic sequences of classical strains are disclosed in U.S. Pat. No. 5,871,744 (Vakharia and Mundt) and EP patent application no. 98201704.8 (Akzo Nobel NV).
In particular, an IBDV mutant according to the invention is provided that comprises part of the VP2 coding sequence of the classical strain D78 encoding a VP2 protein fragment that comprises amino acid positions 253-284 and having the desired codons as defined above.
A preferred IBDV mutant according to the present invention comprises part of the D78 VP2 coding region spanning nucleotide position 884-985 having a nucleotide sequence shown in SEQ ID No. 3. This nucleotide fragment replaces the corresponding fragment of the variant E VP2 coding region and encodes inter alia the following amino acids: 253(His), 254(Gly), 270(Thr) and 284(Thr) (SEQ ID No. 4).
An IBDV mutant according to the present invention may comprise the genetic backbone of the segment A of a variant E IBDV strain, including a mutated VP2 coding region as described above. However, an IBDV mutant according to the invention can also be based on the genetic backbone of another IBDV strain, such as a classical- or GLS strain, a classical strain, more in particular strain D78, being preferred.
In such a xe2x80x9cchimericxe2x80x9d IBDV mutant, VP2 coding sequences on the genetic backbone of segment A of a first type of IBDV strain are replaced by the corresponding, relevant variant E VP2 coding sequences that additionally comprise the desired mutations that are responsible for the advantageous properties of the new IBDV mutant.
The VP2 coding sequences encoding a variant E VP2 protein may comprise the (mutated) genetic information encoding the complete variant E VP2 protein (nucleotide positions 131-1666) or may comprise a fragment thereof that encodes a VP2 protein that is able to induce variant E virus neutralising antibodies. In the latter case the variant E VP2 coding sequences may be complemented with VP2 coding sequences of another IBDV subtype such that the IBDV mutant expresses a complete VP2 protein.
In particular, such an IBDV mutant may comprise at least a (mutated) variant E VP2 coding fragment spanning the so-called variable region of the VP2 gene. Bayliss et al. (J. Gen. Virol. 71, 1303-1312, 1990) determined this region to be located within the nucleotide positions 745-1182.
Preferably, the IBDV mutant comprises the variant E VP2 coding sequences spanning nucleotides 647-1666, including the desired mutations described above.
The generation of the (chimeric) IBDV mutants can be achieved by means of the recently established infectious cRNA system for IBDV (Mundt and Vakharia, Proc. Natl. Acad. Sci. USA 93, 11131-11136, 1996). This reverse genetics system opens the possibility to introduce mutations in the RNA genome of the IBDV. The most important step in this reverse genetics system is to provide full length cDNA clones of the segments A and B of IBD virus, including the nucleotides of the 5xe2x80x2- and 3xe2x80x2- ends of both these segments. After cloning procedures, the full length sequences of segment A and B are operably linked to a promoter which is able to bind a DNA dependent RNA polymerase, such as the T7, SP6 or T3 polymerase, the T7 promoter being preferred. The DNA dependent polymerase is able to describe viral cRNA from full length cDNA clones of segment A and B, respectively. This cRNA is able to induce replication of the virus and the isolation of viable virus. This procedure can be performed with every natural occurring IBDV.
Reverse genetics systems have been described for various IBDV strains, such as D78 (Yao et al., J. Virol. 72, 2647-2657, 1998), strain HK46 (Lim et al., J. Virol. 73, 2854-2862, 1999) and CEF 94 (Boot et al., Virology 265, 330-341, 1999).
The segment B of an IBDV mutant according to the invention can be derived from any IBDV strain, preferably from a classical IBDV strain, most preferably from strain D78 or P2 (U.S. Pat. No. 5,871,744 and EP patent application no. 98201704.8).
IBDV mutants as described above comprising segment B of strain P2 display particular advantageous attenuated and protective properties, especially a mutant having amino 254(Gly) or 254(Gly)/270(Thr).
The desired mutations can be introduced into the IBDV genome by means of methods generally known in the art for this purpose. In particular, the mutation(s) are introduced by means of site-directed mutagenises. Methods for introducing a mutation in the IBDV genome are described herein, but are also generally used in the art (Mundt and Vakharia, 1996, supra; Yao et al., J. Virology 72, 2647-2654, 1998; Mundt et al., 1999, supra; EP patent application no. 98201704.8; Current Protocols in Molecular Biology, eds.: F. M. Ausubel et al., Wiley N.Y., 1995 edition, pages 8.5.1.-8.5.9.and Kunkel et al. in Methods in Enzymology vol. 154, 376-382, 1987).
As demonstrated in the Examples, an IBDV mutant according to the invention displays very favourable properties that can lead to a new type of IBDV vaccine. Therefore, another aspect of this invention is a vaccine for use in the protection of poultry against disease resulting from IBDV infection. The vaccine comprises an IBDV mutant as defined above, together with a pharmaceutical acceptable carrier or diluent.
The IBDV mutant can be incorporated into the vaccine as live attenuated or inactivated virus, however, the live form is preferred because this allows the use of all the advantageous properties of the IBDV mutants of the present invention.
A vaccine according to the invention can be prepared by conventional methods such as for example commonly used for the commercially available live- and inactivated IBDV vaccines. Briefly, a susceptible substrate is inoculated with an IBDV mutant according to the invention and propagated until the virus replicated to a desired infectious titre after which IBDV containing material is harvested.
Every substrate which is able to support the replication of IBDV mutants can be used to prepare the vaccine according to the present invention, including primary (avian) cell cultures, such as chicken embryo fibroblast cells (CEF) or chicken embryo liver cells (CEL), mammalian cell lines such as the VERO cell line or the BGM-70 cell line, or avian cell lines such as QT-35, QM-7 or LMH. Usually, after inoculation of the cells, the virus is propagated for 3-10 days, after which the cell culture supernatant is harvested, and if desired filtered or centrifuged in order to remove cell debris.
Alternatively, the IBDV mutant is propagated in embryonated chicken eggs. In particular, the substrate on which these IBDVs are propagated are SPF embryonated eggs. Embryonated eggs can be inoculated with, for example 0.2 ml IBDV mutant containing suspension or homogenate comprising at least 102 TCID50 per egg, and subsequently incubated at 37xc2x0 C. After about 2-5 days the IBD virus product can be harvested by collecting the embryo""s and/or the membranes and/or the allantoic fluid followed by appropriate homogenising of this material. The homogenate can be centrifuged thereafter for 10 min at 2500xc3x97g followed by filtering the supernatant through a filter (100 xcexcm).
The vaccine according to the invention containing the live virus can be prepared and marketed in the form of a suspension or in a lyophilised form and additionally contains a pharmaceutically acceptable carrier or diluent customary used for such compositions. Carriers include stabilisers, preservatives and buffers. Suitable stabilisers are, for example SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran, glutamate or glucose), proteins (such as dried milk serum, albumin or casein) or degradation products thereof. Suitable buffers are for example alkali metal phosphates. Suitable preservatives are thimerosal, merthiolate and gentamicin. Diluents include water, aqueous buffer (such as buffered saline), alcohols and polyols (such as glycerol).
If desired, the live vaccines according to the invention may contain an adjuvant. Examples of suitable compounds and compositions with adjuvant activity are the same as mentioned below.
Although administration by injection, e.g. intramuscularly, subcutaneously or in ovo of the live vaccine according to the present invention is possible, the vaccine is preferably administered by an inexpensive mass application route commonly used for IBDV vaccination. For IBDV vaccination this route includes drinking water, spray and aerosol vaccination.
For administration by the drinking water route it is customary to deprive the animals of water for about 2 to 4 hours before placing the vaccine containing water in front of them, and it is important that there is enough drinker space for all birds to drink evenly. The vaccine is applied in fresh drinking water at a concentration calculated to give each bird a sufficient dose.
In order to prevent a dramatic reduction of the viable vaccine virus by the presence of small amounts of chlorine, iron, zinc or copper ions in the drinking water, preferably a protectant such as skim milk (powder) is added to the water containing vaccine.
The spray method, comprising the coarse spray and aerosol administration, involves the administration of the live IBDV vaccine incorporated in small liquid particles. In the coarse spray method particles usually have an initial droplet size ranging from 10 to 100 microns and in the aerosol administration method droplets usually range from  less than 1 to 50 microns.
In order to prevent inactivation of the live vaccine virus because of increased concentration of dissolved salts as a result of desiccation of the (tap) water particles, small amounts of a protein protectant, such as skimmed milk, skimmed milk powder or gelatin can be added to aqueous phase.
For the generation of the small particles, conventional spray-apparatus and aerosol generators can be used, such as the commercially available spray generators for knapsack spray, hatchery spray and atomist spray. Also the drinking water vaccination can be carried out using conventional apparatus. Details concerning conventional spray/aerosol- and drinking water vaccination can be found in the xe2x80x9cCompendium, administration of poultry vaccinesxe2x80x9d issued by the Gezondheidsdienst voor Pluimvee, Doorn, The Netherlands, van Eck et al., VI-VII, 1988.
In another aspect of the present invention a vaccine is provided comprising the IBDV mutant in an inactivated form. The major advantage of an inactivated vaccine is the high levels of protective antibodies of long duration that can be achieved against both classical and variant E IBDV strains.
The aim of inactivation of the viruses harvested after the propagation step is to eliminate reproduction of the viruses. In general, this can be achieved by chemical or physical means well known in the art.
A vaccine containing the inactivated IBDV mutant can, for example comprise one or more of the above-mentioned pharmaceutically acceptable carriers or diluents suited for this purpose.
Preferably, an inactivated vaccine according to the invention comprises one or more compounds with adjuvant activity. Suitable compounds or compositions for this purpose include aluminium hydroxide, -phosphate or -oxide, oil-in-water or water-in-oil emulsion based on, for example a mineral oil, such as Bayol F(copyright) or Marcol 52(copyright) or a vegetable oil such as vitamin E acetate, and saponins.
The vaccine according to the invention comprises an effective dosage of the IBDV mutant as the active component, i.e. an amount of immunising IBDV material that will induce immunity in the vaccinated birds against challenge by a virulent virus. Immunity is defined herein as the induction of a significant higher level of protection in a population of birds after vaccination compared to an unvaccinated group.
Typically, the live vaccine according to the invention can be administered in a dose of 102-109 TCID50 infectious dose50 (TCID50) per animal, preferably in a dose ranging from 104.0-107.0 TCID50. Inactivated vaccines may contain the antigenic equivalent of 105-109 TCID50 per animal.
Inactivated vaccines are usually administered parenterally, e.g. intramuscularly or subcutaneously.
Although, the IBDV vaccine according to the present invention may be used effectively in chickens, also other poultry such as turkeys, guinea fowl and partridges may be successfully vaccinated with the vaccine. Chickens include broilers, reproduction stock and laying stock.
The age of the animals receiving a live or inactivated vaccine according to the invention is the same as that of the animals receiving the conventional live- or inactivated IBDV vaccines. For example, broilers (free of maternally derived antibodies-MDA) may be vaccinated at one-day-old or in ovo, whereas broilers with high levels of MDA are preferably vaccinated at 2-3 weeks of age. Laying stock or reproduction stock with low levels of MDA may be vaccinated at 1-10 days of age followed by booster vaccinations with inactivated vaccine on 6-12 and 16-20 weeks of age.
The invention also includes combination vaccines comprising, in addition to the IBDV mutant described above, one or more immunogens derived from other pathogens infectious to poultry.
Preferably, the combination vaccine additionally comprises one or more vaccine strains of Mareks Disease virus (MDV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), egg drop syndrome (EDS) virus, turkey rhinotracheitis virus (TRTV) or reovirus.